Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic -cells triggered by complex interactions of environmental factors in genetically predisposed individuals. Previous studies carried out in this laboratory revealed a panel of novel peptides and corresponding proteins having very good discriminating power of T1D from healthy controls using liquid chromatography-mass spectrometry (LC-MS) based proteomics analyses of human blood serum and plasma samples from a Diabetes Antibody Standardization Program cohort with limited sample size. The long-term goal of this project is to provide thoroughly validated diagnostic and prognostic markers to the clinical community for intervention of T1D, and to understand the pathogenesis of T1D for its ultimate prevention. In the present application, using The Diabetes Autoimmunity in the Young Study (DAISY) as a parent T1D cohort, we will use our multiplexed liquid chromatography- multiple reaction monitoring-mass spectrometry (LC-MRM-MS) platform and state-of-the-art technologies on intact protein separation coupled with high resolution tandem mass spectrometry to measure these biomarkers in large numbers (n = 2090) of blood serum/plasma from independent, large scale T1D cohorts, as well as from individuals having diseases that share similar clinical and immunological outcomes with T1D. Specifically, we propose the following aims: Aim 1, to establish the sensitivity of marker peptides in an independent T1D cohort and provide accurate threshold values of these peptides in diagnosis of T1D; Aim 2, to establish the specificity of marker peptides using disease controls of type 2 diabetes, celiac disease and inflammatory bowel disease; Aim 3, to identify the protein isoforms of key marker peptides having potential roles in pathogenesis of this disease; Aim 4, to validate the peptide biomarkers in large scale clinical T1D cohorts blinded to the investigators; and Aim 5, to establish the prognostic value of validated peptide biomarkers with longitudinal samples from the DAISY cohort. The outcome of the proposed research will confirm the utility and specificity of these peptide/protein markers for diagnosing T1D, gain further insight to the pathogenesis of this disease, and provide foundations for new strategies in T1D prognosis, intervention and prevention.